It has been hotly debated whether multiple items in working memory are represented simultaneously or sequentially. A recent study found that items in different sequence positions elicited gamma power that is phase locked to distinct phase of a theta oscillation, supporting the sequential representation model. However, the results are based on activities in encoding period and it still remains unknown how the sequentially memorized items are represented during maintaining period. In the present study, we recorded EEG activities while human subjects performed a sequential working memory task. In each trial, subjects were first presented with multiple rectangles with different orientation and color, and were instructed to only memorize the orientation of the cued rectangles and their temporal order ("Encoding phase"). Next, they performed a central fixation task, and were simultaneously presented with circles either endowed with memory-related color or memory-unrelated color ("Maintaining phase"). Finally, they were asked to judge whether or not the orientation of a presented rectangle was similar to that of the to-be-memorized rectangles in the Encoding phase ("Recalling phase"). Critically, we employed a temporal response function technique (TRF) to extract item-specific response in "Maintaining phase". We found that first, the TRF responses for the to-be-memorized items exhibited stronger alpha-band (~10 Hz) power compared to non-memorized item. Second, the alpha power profiles for the multiple to-be-memorized items showed a time-reversed alpha activation pattern. Specifically, the item that occupied earlier (later) sequential position in Encoding phase elicited later (earlier) alpha responses in Maintaining period. Finally, the sequential activation sequence became faster as the memory list became longer. In summary, our results support the sequential representation model in working memory, and provide direct neuronal evidence in human subjects that sequential memory is mediated by re-activating sequences in a time-reversed manner during maintaining period.